Membrane Protein Solid State NMR: PISEMA Development and the M2 Tetramer Structure

膜蛋白约占所有基因组的30%。今天，蛋白质数据库中只有不到40种独特的膜蛋白结构。膜蛋白对细胞执行许多关键功能，它们与水溶性蛋白有很大的不同：它们的氨基酸组成，稳定它们结构的力量，它们的内部动力学等。该技术独特地利用完全水合的脂质双层来溶解蛋白质样品。这项工作将进一步发展用于结构表征的主要固态NMR实验，PISEMA。一些修改，包括在李-戈德堡序列期间使用斜坡脉冲，表明在使PISEMA成为更稳健的实验方面有显着的收益。NHMFL提供的高场磁体中具有平衡RF电路的先进NMR探头将极大地帮助实现这一目标。对α-螺旋蛋白质的PISEMA光谱的分析导致了《磁共振杂志》和《蛋白质科学》的封面报道。固态NMR数据的表征直接导致脂质双层内的螺旋的倾斜和旋转取向的描述。在这里，这种分析将使用数学表达式来显示如何在结构解决方案中的简并性，通过显示独特的光谱模式预测的α，310，和π螺旋，并通过表征更复杂的光谱模式的螺旋在平面的脂质双层。此外，这是该项目的目的，以解决完整的骨干结构和组装的44 kDa的四聚体M2质子通道在水合脂质双层以上的脂质相变。 在这个项目中，Cross博士和他的同事们将开发一种独特的方法来确定膜蛋白在其天然环境中的三维结构-围绕每个细胞的脂质膜。这些蛋白质控制着进入和离开细胞的一切，它们负责在细胞之间接受和发送信息。给定基因组的30%或更多的蛋白质是膜蛋白，然而技术对于它们的结构表征非常有限。Cross博士拥有一支由化学家、数学家和生物化学家组成的独特团队，汇集了开发核磁共振方法的必要技能，该方法现已被证明可实现膜蛋白的独特结构。克罗斯博士和他的同事们正在完善这项技术，提高其灵敏度，以适应更广泛的应用。这项研究将对膜蛋白生物物理学和结构生物学领域产生广泛影响，化学、生物和数学学科的学生将在经过验证的跨学科环境中接受培训。

Membrane proteins represent approximately 30% of all genomes yet characterized. Today, less than 40 unique membrane protein structures are in the Protein Data Bank. Membrane proteins carry out numerous critical functions for cells and they are vastly different from their water-soluble counterparts through: their amino acid composition, the forces that stabilize their structure, their internal dynamics, etc. Sold state NMR is a demonstrated technology for achieving 3D high resolution structure in a lipid bilayer environment. This technology uniquely utilizes fully hydrated lipid bilayers for solvating protein samples. This work will further develop the primary solid state NMR experiment used for the structural characterization, PISEMA. Several modifications including the use of ramped pulses during the Lee-Goldburg sequence suggests significant gains in making PISEMA a more robust experiment. Advanced NMR probes with balanced RF circuits in high field magnets available at the NHMFL will be a great help in achieving this goal. The analysis of PISEMA spectra of alpha-helical proteins has led to cover stories in both the Journal of Magnetic Resonance and Protein Science. The characterization of solid state NMR data leads directly to a description of the tilt and rotational orientation of the helix within the lipid bilayer. Here, this analysis will be advanced using mathematical expressions to show how degeneracies in the structural solutions are resolved, by showing that unique spectral patterns are predicted for alpha, 310, and pi helices, and by characterizing the more complex spectral patterns for helices in the plane of the lipid bilayer. Moreover, it is the aim of this project to solve the complete backbone structure and assembly of the 44 kDa tetrameric M2 proton channel in hydrated lipid bilayers above the phase transition of the lipids. In this project Dr. Cross and his colleagues will develop a unique approach for determining the three dimensional structure of membrane proteins in their native environment - the lipid membrane that surrounds each cell. These proteins control everything that enters and leaves the cells, and they are responsible for accepting and sending information between cells. 30% or more of the proteins of a given genome are membrane proteins and yet technologies are very limited for their structural characterization. With a unique team of chemists, mathematicians and biophysicists, Dr. Cross brings together the necessary skills for the development of a Nuclear Magnetic Resonance methodology that has now been demonstrated to achieve unique structures of membrane proteins. Dr. Cross and his coworkers are refining the technology and enhancing its sensitivity for greater applications. This research will have a broad impact in both the fields of membrane protein biophysics and structural biology and students in chemical, biological and mathematical disciplines will be trained in a proven interdisciplinary environment.